Therapeutic uses of dunaliella powder

ABSTRACT

A method for treating a disease selected from diabetes mellitus and atherosclerosis, and a method for reducing triglycerides and/or increasing HDL cholesterol levels in the plasma of a subject. The method comprises administrating. to a subject an effective amount of crude  Dunaliella  powder, optionally together with an activator of nuclear receptors.

FIELD OF THE INVENTION

This invention relates to the use of crude Dunaliella bardawil powder inthe treatment of a number of conditions including high triglyceride andlow high-density lipoprotein (HDL) cholesterol levels, atherosclerosisand diabetes.

REFERENCES

The following is a list of references believed to be pertinent as abackground to the present invention:

-   -   1. Ben-Amotz, A. and Shaish, A., Carotene Biosynthesis in        Dunaliella: Physiology, Biochemistry and Biotechnology, Ed.        Avron, M. and Ben-Amotz., 9:206-16, 1992.    -   2. U.S. Plant Pat. No. 4,511, issued Mar. 18, 1980.    -   3. U.S. Pat. No. 4,199,895, issued Apr. 29, 1980.    -   4. Ben-Amotz, A., Mokady, S., Edelstein, S. and Avron, M.,        Bio-availability of a natural isomer mixture as compared with        synthetic all-trans beta-carotene in, rats and chicks, J.        Nutrition, 119:1013-1019, 1989.    -   5. Day, C., Thiazolidinediones: a new class of antidiabetic        drugs Diabetic Med. 16:179-192, 1999.    -   6. Mukherjee, R., Davies, P. J. A. et al., Sensitization of        diabetic and obese mice to insulin by retinoid X receptor        agonists. Nature, 386:407-410, 1997.

BACKGROUND OF THE INVENTION

Two strains of Dunaliella, a unicellular, biflagellate, wall-less greenalga, are capable of producing very large amounts of β-carotene,Dunaliella salina Teod. and Dunaliella bardawil ⁽¹⁾ . D. bardawil is ahalotolerant alga whose β-carotene content comprises about 50%all-trans-β-carotene with the remainder composed mostly of 9-cisβ-carotene and a few other β-carotene isomers⁽²⁾. A process has beendescribed for cultivating D. bardawil so as to obtain algae containingup to about 5% by weight of β-carotene⁽³⁾. Later developments of theprocess increased the percentage to more than 8%. It has been shown thatthe natural isomer mixture of β-carotene which is accumulated in thealga Dunaliella bardawil is accumulated in fatty tissues of rats andchicks to an extent which is about 10 fold higher than that observed byfeeding the synthetic all-trands β-carotene⁽⁴⁾.

Various carotenoid-enriched Dunaliella commercial products are availablesuch as Betatene™ (produced by the Henkel Corp., Germany) and Nutrilite™(Amway, Inc., U.S.A.). These products are oil extracts of carotenoidsfrom, Dunaliella.

Retinoids are essential for a living organism for vision, cellulargrowth, differentiation, and to maintain the general health of theorganism. 9-cis-retinoic-acid and all-trans-retinoic-acid are producedin the body by the cleavage of β-carotene (BC). The retinoid nuclearreceptors, also known as orphan receptors RAR and RXR, have distinctphysiologic properties by activating transcriptional. factors. All-transretinoic-acid binds to RAR but not to RXR, while 9-cis retinoic-acidbinds to RXR, which plays a key role in important biological processes.

The activation of nuclear receptors is essential to cell metabolism.,especially with respect to lipids and glucose. Several nuclear receptorshave been implicated in cholesterol homeostasis. These receptorsinclude: the liver X receptors (LXRa/NR1H3 and LXRb/NR1H2) and farnesoidX receptor (FXR/NR1H4) that are bound and activated by oxysterols andbile acids, respectively. LXR and FXR form obligate heterodimters withRXR that is activated by 9-cis retinoic acid and synthetic agonists(termed rexinoids). It was recently found that the activation of RXR byits ligands can affect two central processes of cholesterol metabolism:I. Cholesterol absorption in the intestine(activation of RXR/FXRheterodimer repressed cholesterol 7α-hydroxylase (CYP7A1) expression,resulting in decreased bile acid synthesis and cholesterol absorption).II. Reverse cholesterol transport from peripheral tissues (activation ofRXR/LXR heterodimer inhibited cholesterol absorption and induced reversecholesterol, transport in peripheral tissues).

The following nuclear receptors are known to form heterodimers with RXRand hence, can be potentially activated by 9-cis retinoic acidadministration: thyroid hormone (TRa/b); vitamin, D (VDR); fattyacid/eicosanoids (PPARα/βγ); oxysterols (LXRa/b); bile acids (FXR);pregnanes/bile acids/xenobiotics (PXR); androstanes/xenobiotics (CAR).

Fibrates (clofibrate, fenofibrate, bezafibrate, ciprofibrate,beclofibrate and gemfibrozil) are currently recommended for thetreatment of patients with hypertriglyceridemia (high plasma TG). Thetreatment of hypertriglycerdemia results in decreased plasma levels oftriglyceride-rich lipoproteins. HDL cholesterol levels are usuallyincreased when the baseline levels are low. The increase in HDLcholesterol is usually concomitant with, increased levels of the HDLapolipoprotein A-I and A-II. In addition, fibrates reduce theatherogenic apoC-III-containing particles, effect post-prandial lipemiaand some fibrate lower plasma fibrinogen and CRP levels.

As a consequence of their effect on lipid metabolism, fibrates have beenshown to affect specific lipoprotein disorders such as in combinedhyperlipidemia, primary hypertriglyceredemia, type IIIdysbetalipoprotenemia, and non-insulin dependent diabetes mellitus(NIDDM).

Fibrates are considered to be well tolerated, with an excellent safetyprofile. A low toxicity has been, reported in almost every organ system.Fenofibrate treatment revealed low frequency of side effects. Long-termadministration revealed no effect on peroxisome proliferation in humanliver and no evidence for carcinogenesis. Clinically relevantinteraction of fibrates with other anti-hyperlipedemic drugs includerhabdomyolysis and decreased bioavailibility when combined with somebile acid sequestrants. Potentiation of the anticoagulant effect ofcoumarin may cause bleeding.

Fibrates mediate at least part of their effects by peroxisomeproliferator-activated receptor α (PPAR-α). Upon activation withfibrates, PPARs heterodimerize with another nuclear receptor, the 9-cisretinoic acid receptor (RXR). The dimer binds to specific responseelements, termed peroxisome proliferator response elements (PPREs) andregulates gene expression.

Low plasma levels of high-density lipoprotein (HDL) and hightriglyceride (TG) plasma levels are risk factors for atherosclerosis.Low levels of plasma HDL cholesterol, apolipoprotein AI (apoA-T) andhigh levels of triglycerides (TG) are associated with increased risk foratherosclerosis, the major cause of morbidity and mortality in Westernsocieties. A recent study showed that the rate of coronary events isreduced by 22 percent by raising HDL cholesterol levels and lowering theplasma TG levels in patients treated with the fibrate gemfibrozil.

Several modes of action were proposed for the fibrate beneficial effectson atherosclerosis: Induction of lipoprotein lipolysis by reducingapoC-III levels and/or by increasing lipoprotein lipase activity.Induction of hepatic fatty acid (FA) uptake by the induction ofFA-transporter protein and acyl-CoA synthetase. Decrease apoB and VLDLproduction. Reduction of hepatic TG production by induction ofperoxisome (in rodents only) or mitochondrial β-oxidation pathway andinhibition of FA synthesis. Most important, fibrates increase theproduction of apoA-I and apoA-II in the liver, which probably contributeto the process of reverse cholesterol transport.

Type 2 diabetes mellitus is a serious health problem. It arises when,resistance to the glucose lowering effects of insulin combines withimpaired insulin secretion to raise the levels of glucose in the bloodbeyond the normal range. Thiazolidinediones are a new class ofantidiabetic agents that improve insulin sensitivity and reduce plasmaglucose and blood pressure in subjects with type 2 diabetes⁽⁵⁾. Thedrugs bind and activate PPARγ that binds to DNA as heterodimers with acommon partner, retinoid X receptor (RXR) to regulate transcription. RXRagonists have been shown to function as insulin sensitizers, markedlydecreasing serum glucose, triglycerides and insulin in obese mice⁽⁶⁾.

WO 93/24454 describes a carotenoid composition derived from Dunaliellaalgae in which the β-carotene content is predominantly 9-cis β-carotene.There is no mention of any medical applications.

U.S. Pat. No. 5,219,888 (Katocs) discloses a method to increase plasmaHDL levels for the treatment and prevention of coronary artery diseaseby administrating a therapeutic amount of the retinoids alltrans-retinoic acid and 9-cis-retinoic acid.

U.S. Pat. No. 5,948,823 discloses use of a substantially crudeDunaliellia algae preparation in protecting mammals against thedetrimental effects of medical and nuclear irradiation.

WO 97/10819 teaches a new treatment for non-insulin dependent diabetesmellitus by administration of RXR agonists and optionally, peroxisomeproliferation activated receptor gamma agonists.

WO 99/50658 identifies compounds which modulate nuclear receptoractivity, used for treating e.g. cancer, cardiovascular disease,osteoporosis, diabetes, postmenopausal disorders and inflammatoryconditions.

WO 2001119770 reveals new retinoid X analogs, useful as retinoid Xreceptor modulators for lowering blood glucose levels, for modulatinglipid metabolism and in treatment of e.g. diabetes, obesity,cardiovascular diseases and breast cancer.

U.S. Pat. Nos. 5,972,881 and 6,028,052 (Heyman) disclose methods andcompositions for the treatment of non-insulin dependent diabetes usingan RXR agonist together with a PPARγ agonist.

WO 03/027090 provides novel retinoid compounds that have selectivity asRXR agonists, and are effective in reducing blood glucose andmaintaining body weight, thus being useful for the treatment of diabetes(NIDDIM) and obesity.

Colagiuri S. and Best, J. Lipid-lowering therapy in people with type 2diabetes. Current Opinion in Lipidology (2002) 13:617-623, teaches thatlipid powering with statins and fibrates is effective in improvingcardiovascular disease outcome in diabetes.

Levy, Y. et al. Dietary supplementation of a natural isomer mixture ofBeta-carolene inhibits oxidation of LDL derived from patients withdiabetes mellitus. Nutrition & Metabolism (2000) 44:54-60, describesexperiments in which Dunaliella bardawil-derived β-carotene wassupplemented to the diet of diabetic patients. The dietarysupplementation normalized the enhanced LDL susceptibility to oxidationin these patients.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for treatingpatients suffering from diabetes, low plasma HDL and/or high plasma TGand/or atherosclerosis.

The present invention provides by one of its aspects, a method fortreating a disease selected from diabetes mellitus and atherosclerosiscomprising administrating to a subject an effective amount of crudeDunaliella powder.

The present invention also provides by another aspect a method forreducing triglycerides and/or increasing HDL cholesterol levels in theplasma of a subject comprising administrating to the subject aneffective amount of crude Dunaliella powder.

In accordance with another aspect of the invention there is provided amedicament for use in treating diabetes mellitus, low plasma HDL and/orhigh plasma TG and/or atherosclerosis, comprising an effective amount ofa substantially crude Dunaliella algae preparation.

In accordance with a further aspect of the invention, there is providedthe use of an effective amount of a substantially crude Dunaliella algaepreparation in, the preparation of a pharmaceutical composition for usein treating diabetes mellitus, low plasma HDL and/or high plasma TGand/or atherosclerosis

The active ingredient in accordance with the invention is asubstantially crude Dunaliella algae preparation, typically driedDunaliella algae. The Dunaliella algae are preferably Dunaliellabardawil.

The terms “heating” or “treatment” in the present specification shouldbe understood as including both the curing of the disease, as well asbringing about an improvement in the pathological symptoms of thedisease. The terms may also include prevention of the diseases. Theseterms also relate to the potential to inhibit both the initiation andprogression of atherosclerosis and to the reduction of inflammation.,thereby inhibiting and preventing complications of the disease.

An “effective amount” should be understood as an amount or dose of theactive ingredient which is sufficient to achieve the desired therapeuticeffect, i.e. treatment of the indicated diseases. The effective amountdepends on various factors including the severity of the disease, theadministration regimen, e.g. whether the preparation is given once orseveral times over a period of time, the physical condition of thesubject; etc. The artisan should have no difficulties, by minimalexperiments, to determine the effective amount in each case.

The term “atherosclerosis” includes all types of the disease includingcalcified plaques, non-calcified plaques, fibrocalcified plaques andothers. The term “diabetes” includes all types of diabetes, inparticular type 2 diabetes mellitus.

In a preferred embodiment, the crude Dunaliella powder is administeredtogether with one or more activators of nuclear receptors. Theactivators of nuclear receptors are preferably peroxisomeproliferator-activated receptor α and γ (PPARα and PPARγ) agonists, suchas fibrates and thiazolidinediones. Non-limiting examples of fibratesare clofibrate, fenofibrate, bezafibrate, ciprofibrate, beclofibrate andgemfibrozil. Non-limiting examples of thiazolidinediones are avandia,troglitazone, BRL 49653, pioglitazone, ciglitazone, WAY-120,744,englitazone, AD 5075, darglitazone and rosiglitazone.

The crude Dunaliella powder is preferably administered orally, forexample in an encapsulated form.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in, practice, a preferred embodiment will now be described, by wayof non-limiting example only, with reference to the accompanyingdrawings, in which:

FIG. 1 illustrates the effect of a combination treatment of fibrates andcrude Dunaliella powder on plasma HDL cholesterol levels in human apoAItransgenic mice.

FIGS. 2-4 show the effect of crude Dunaliella powder on atherogenesis bythe size (area) of atherosclerotic lesions at the aortic sinus in maleapoE-deficient mice (FIGS. 2 & 3) and in male LDL receptor-deficientmice (FIG. 4);

FIG. 5 illustrates measurement of cholesterol absorption inapoE-deficient mice;

FIG. 6 shows HDL-cholesterol levels in patients receiving a dualtreatment of fibrate with and without crude Dunaliella powder; and

FIGS. 7-10 show plasma insulin, plasma glucose plasma triglycerides andplasma cholesterol levels in male LDL R−\− mice with induced diabetes.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Methods and Materials

All of the human studies described below employed capsules containingDunaliella powder prepared as follows.

Dunaliella bardawil (hereinafter “Db”) was grown and cultivated in largebody open salt water ponds of 50,000 m² to obtain algae comprising 8% byweight of β-carotene (hereinafter “BC”) at an approximately 1:1 ratio ofall-trans and 9-cis BC (4,9). The algae were harvested by dislodgingcentrifuges into a concentrated paste. The paste was washed to removethe salt and sterilized, and then spray dried to yield Db powdercomprising approximately 8% BC and less than 5% moisture. The powder waspackaged in capsules of 250 mg algae containing 20 mg of BC eachtogether with all of the natural components of the algae. The BC of thecapsules retains the original 1:1 ratio of isomers. The capsules arepackaged in vacuum closed blisters which have a shelf life of up tothree years. For the animal studies the Db powder was mixed with thediet to give 8% of Dunaliella powder in the feed.

EXAMPLE I

The aim of the first experiment was to study the effect of acombination, treatment of fibrates and a crude Dunalielia powder-richdiet on plasma apoAI and HDL cholesterol levels.

Six month old male littermates, transgenic human apoAI mice on thebackground of C57BL/6 mice were used (Rubin E M, Ishida B Y, Cliff S M,Krauss R M. (1991) Proc Natl Acad Sci USA. January 15;88(2):434-8,Expression of human apolipoprotein A-I in transgenic mice results inreduced plasma levels of murine apolipoprotein A-I and the appearance oftwo new high density lipoprotein size subclasses). The mice contain theliver-specific enhancer of the human apo A-I gene promoter necessary todrive hepatic apo A-I expression. The mice (n=24) were divided into fourgroups (n=6 in each group) and fed daily with a standard diet (Koffolk,Israel Diet no.19510) ad libitum. The first group (control) wasadministrated with phosphate buffered saline (PBS). The second group(Fibrate) was administrated with ciprofibrate (Lipanor, CTZ, Israel), 30mg/Kg body weight, in PBS. The third group (Dunaliella) was supplementedwith. PBS and crude Dunaliella powder. The fourth group was supplementedwith ciprofibrate and crude Dunaliella powder, which provided ˜4 g of9-cis-β-carotene per one Kg of the diet. The mice were sacrificed, after4 weeks of treatment.

The results (FIG. 1) showed that plasma HDL cholesterol levels reachedthe highest levels in mice treated with both fibrate and crudeDunaliella powder (p<0.05 all pairwise multiple comparison procedures,“control” group versus “ciprofibrate plus crude Dunaliella powdergroup”). The lowest level was detected in the control group, 408±42mg/dl, and the highest in the combination of ciprofibrate and crudeDunaliella powder treated group, 535±71 mg/dl. A similar trend, althoughnot statistically significant, was observed in apoaI plasma levels.

EXAMPLE II

To test the effect of Dunaliella on atherogenesis, male apoE-deficientmice (n=10) were fed with standard chow diet as defined above,supplemented with 8% of crude Dunaliella powder for 6 weeks. A controlgroup (n=10) received chow diet alone. Atherosclerosis, determined bythe lesion size at the aortic sinus, was 34% lower in the group of micetreated with crude Dunaliella powder than in the control group of mice(FIG. 2).

EXAMPLE III

To test the effect of Dunaliella powder on atherogenesis, maleapoE-deficient mice (n=13) were fed a standard chow diet supplementedwith 8% Dunaliella powder for 8 weeks. A second group (n=12) was treatedwith oxidized Dunaliella powder, containing no β-carotene, while thecontrol group (n=12) received chow diet alone. Atherosclerosis,determined by the lesion size at the aortic sinus, was 33% lower inDunaliella treated group than in control group, p=0.027 (FIG. 3). Incontrast, oxidized Dunaliella powder did not affect atherogenesis.

EXAMPLE IV

To test the effect of Dunaliella on atherogenesis, male LDLreceptor-deficient mice (n=10) were fed a standard chow dietsupplemented with 8% Dunaliella powder for 3 weeks followed by 7 weeksof Western diet. A second group was treated with oxidized Dunaliellapowder (n=10) and control group (n=10) received chow diet followed byWestern diet, alone. Atherosclerosis, determined by the lesion size atthe aortic sinus, was 65% lower in Dunaliella treated group than incontrol group, p=0.004 (FIG. 4). In contrast, oxidized Dunaliella powderdid not affect atherogenesis.

EXAMPLE V

Cholesterol absorption was measured by a fecal dual-isotope method,which measures the ratio of fecal excretion of ³H-sitostenol (which isnot absorbed) to ¹⁴C-cholesterol (which is variably absorbed). TheapoE-deficient mice were divided into 2 groups, the group of micetreated with Dunaliella was administrated with 8% of crude Dunaliellapowder in chow diet for 3 weeks (n=6) and a control group (n=6) receivedchow diet alone. Administration of crude Dunaliella powder toapoE-deficient mice resulted, (similarly to previously described RXRagonists) in inhibition of cholesterol absorption. (FIG. 5).

EXAMPLE VI

The aim of the human trial was to study the effect of the combination offibrate and crude Dunaliella bardawil powder in patients with low HDL(<35 mg/dl) and high TG levels (>250 mg/dl).

At the first stage the patients (n=20) received fibrates (ciprofibrateor bezafibrate) for at least 3 months. At the second stage of the studythe patients received 4 pills of crude Dunaliella powder according tothe invention, 2 pills with breakfast and 2 pills with dinner, toprovide a total daily uptake of 80 mg of β-carotene. Plasma lipidprofile was detected after each stage of the study.

The results (FIG. 6) show that plasma HDL levels were significantly(p=0.002, paired. t-test) higher after the combined treatment of fibrateand crude Dunaliella powder treatment in comparison to fibrate alone(29.3±6.8 mg/dl versus 36.5±9.9 mg/dl, respectively; a 24.5% increase).

A similar trend, although not significant (p=0.35) was observed inplasma apoAI levels, 120.9±15.2 versus 128.4±28.2 in fibrate and fibratetogether with β-carotene, respectively. These findings suggest that thecombined treatment affects both apoAI synthesis and the process ofreverse cholesterol transport.

EXAMPLE VII

To test the effect of Dunaliella on Diabetes, male LDL R−\− mice (n=30)were fed a Western diet (42% calories from fat) in order to induceDiabetes. After 4 weeks of Western diet feeding, the mice were dividedinto 6 groups (n=5 in each group) and were fed Western diet fortified byrosiglitazone (0.02% of the diet, weight/weight), crude Dunaliellapowder (8% of the diet, weight/weight) or 9-cis retinoic acid (24nmol/mouse×day). The treatments lasted 4 weeks.

The 8% Dunaliella powder provided ˜1.5 nmol. 9-cis retinoicacid/mouse×day. This calculated amount is based on conversion ofβ-carotene to vitamin A (27 molecules provide 1 molecule of vitamin Aand on the ratio of retinoic acid to vitamin A which is about 1 moleculeof retinoic acid to 300 molecules of retinol, retinal and retinoicacid).

The following groups were included:

-   -   1. Control, Western diet alone. (Control)    -   2. 9-cis retinoic acid (RA)    -   3. Rosiglitazone (Rosi)    -   4. Dunaliella powder 8% (Dun.)        Results

Dunaliella treatment reduced both plasma glucose and insulin levels ascompared to rosiglitazone and control groups (FIGS. 7 and 8). Moreover,Dunaliella treatment reduced plasma cholesterol and TG levelssignificantly comparing to rosiglitazone group (FIGS. 9 and 10). On theother hand, 9-cis retinoic acid did not affect plasma insulin andglucose levels and their levels in Dunaliella group were significantlylower. The results demonstrate that Dunaliella powder can reduce plasmainsulin and glucose levels more efficiently than 9-cis retinoic acid orrosiglitazone in LDLR−\− mouse model.

1. A method for treating a disease selected from diabetes mellitus andatherosclerosis comprising administrating to a subject an effectiveamount of crude Dunaliella powder comprising an approximately 1:1 ratioof all-trans and 9-cis β-carotene.
 2. A method for reducingtriglycerides and/or increasing HDL cholesterol levels in the plasma ofa subject comprising administrating to the subject an effective amountof crude Dunaliella powder.
 3. The method according to claim 1 whereinsaid crude Dunaliella powder is administered together with one or moreactivators of nuclear receptors.
 4. The method according to claim 3wherein the activators of nuclear receptors are peroxisomeproliferator-activated receptor α or γ (PPARα or PPARγ) agonists.
 5. Themethod according to claim 4 wherein the PPARα or PPARγ agonists areselected from fibrates and thiazolidinediones.
 6. The method accordingto claim 5 wherein the fibrates are selected from clofibrate,fenofibrate, bezafibrate, ciprofibrate, beclofibrate and gemfibrozil. 7.The method according to claim 5 wherein the thiazolidinediones areselected from, troglitazone, BRL 49653, pioglitazone, ciglitazone, WAY120,744, englitazone, AD 5075, darglitazone and rosiglitazone.
 8. Themethod according to claim 1 wherein said crude Dunaliella powder isadministered orally.
 9. The method according to claim 1 wherein saidDunaliella is Dunaliella bardawil.
 10. The method according to claim 1,wherein said powder is encapsulated.
 11. The method according to claim 2wherein said crude Dunaliella powder is administered together with oneor more activators of nuclear receptors.
 12. The method according toclaim 11 wherein the activators of nuclear receptors are peroxisomeproliferator-activated receptor α or γ (PPARα or PPARγ).
 13. The methodaccording to claim 12 wherein the PPARα or PPARγ agonists are selectedfrom fibrates and thiazolidinediones.
 14. The method according to claim13 wherein the fibrates are selected from clofibrate, fenofibrate,bezafibrate, ciprofibrate, beclofibrate and gemfibrozil.
 15. The methodaccording to claim 13 wherein the thiazolidinediones are selected fromtroglitazone, BRL 49653, pioglitazone, ciglitazone, WAY 120,744,englitazone, AD 5075, darglitazone and rosiglitazone.
 16. The methodaccording to claim 2 wherein said crude Dunaliella powder isadministered orally.
 17. The method according to claim 2 wherein saidDunaliella is Dunaliella bardawil.
 18. The method according to claim 2,wherein said powder is encapsulated.